Molecular mechanisms of potassium channel permeation and gating

 描述(申请人提供)：钾(K+)通道是细胞兴奋性的主要决定因素，在许多生理过程中发挥关键作用。对离子通道的机械理解的核心是门控过程，即通向离子流的孔洞的打开和关闭。门控故障可能会造成灾难性的生理后果。这项资助的总体目标是通过对原核K+通道模型、真核K+通道的同源物进行功能、结构和理论分析，了解K+通道中配体调节和门控的分子机制。细菌通道有助于对这些机制进行必要的生化和结构研究。我们的第一个主要目标是以KCSA为模型来理解K+通道中的配基门控。在AIMS 1.1和1.2中，我们建议使用核磁共振光谱直接确定两个质子敏感残基的pKas(本征质子结合亲和力)，无论是在开放状态还是在闭合状态。我们将利用电生理学确定的改变pH门控的突变体来指导对核磁共振谱中峰分配和化学位移变化的解释。我们还将使用交联的封闭和开放的KCSA结构，这将使我们能够在没有通道门的构象变化的情况下确定pH敏感残基的pKA。据我们所知，这将是第一次直接确定开态和闭合状态的固有配体结合亲和力。在目标1.3中，我们将建立一个用于KCSA质子门控的Monod-Wyman-Changeux模型，该模型结合了建模、诱变、电生理学、X射线结晶学和核磁共振的结果。X射线结晶学将被用来从结构上确定通道突变的影响。我们的第二个主要目标是以MthK为模型来理解钙激活的K+通道的门控。AIMS 2.1和2.2研究了受钙离子调制的门的物理位置。我们将通过确定与K+通道前庭(两个门之间)结合的细胞内阻滞剂的通道是否取决于通道是开放的还是关闭的，来调查MthK钙激活门是在束交叉还是在选择性过滤器处。在没有钙离子的情况下，阻滞剂对前庭的可及性是选择性过滤门的标志。在目标2.3中，我们建议通过改变双层组成来研究MthK缓慢脱敏，这种现象只在脂质体中观察到，而在双层记录中没有观察到。这种现象很耐人寻味，因为它的不同出现将表明基本的、生理上相关的通道属性是由双层参数数字调制的。我们将使用停流散装实验，测量荧光团通过MthK重组囊泡进入Tl+时的猝灭情况，以确定阻滞剂在存在和不存在钙离子的情况下的亲和力和动力学，并确定脂类对脱敏的影响。所提出的目标将为我们提供对K+通道基本特性的新见解，这将很容易适用于我们对更广泛的真核K+通道家族的理解。

 DESCRIPTION (provided by applicant): Potassium (K+) channels are major determinants of cell excitability and play crucial roles in many physiological processes. Central to a mechanistic understanding of ion channels is the process of gating, the opening and closing of the pore to ionic flow. Gating malfunctions can have disastrous physiological consequences. The overall objective of this grant is to understand the molecular mechanisms of ligand modulation and gating in K+ channels by employing functional, structural, and theoretical analysis on model prokaryotic K+ channels, homologues of eukaryotic K+ channels. Bacterial channels lend themselves to the biochemical and structural studies necessary to investigate these mechanisms. Our first major aim is to understand ligand gating in K+ channels using KcsA as a model. In Aims 1.1 and 1.2 we propose to determine directly the pKas (the intrinsic proton binding affinities) of two proposed proton-sensing residues using NMR spectroscopy, in both open and closed states. We will take advantage of mutants with changed pH gating as determined with electrophysiology to guide interpretations of peak assignments and chemical shift changes in the NMR spectra. We will also use crosslinked closed and open KcsA constructs that will allow us to determine pKas of pH sensing residues in the absence of the conformational change that gates the channel. To our knowledge, this would be the first direct determination of intrinsic ligand binding affinities to both open and closed states. In Aim 1.3 we will formulate a Monod-Wyman-Changeux model for proton gating in KcsA constrained with results from the combined approach of modeling, mutagenesis, electrophysiology, X-ray crystallography and NMR. X-ray crystallography will be used to structurally determine the effect of channel mutations. Our second major aim is to understand gating of Ca2+-activated K+ channels using MthK as a model. Aims 2.1 and 2.2 investigate the physical location of the gate modulated by Ca2+. We will investigate whether the MthK Ca2+ activation gate is at the bundle crossing or at the selectivity filter by determining if the access of intracellular blockers that bnd inside the vestibule of K+ channels (between the two gates) depends on whether the channel is open or closed. Accessibility of the blockers to the vestibule in the absence of Ca2+ is an indication of a selectivity filter gate. In Aim 2.3 we propose to investigate MthK slow desensitization, a phenomenon observed only in liposomes, not in bilayer recordings, by altering the bilayer composition. This phenomenon is intriguing as its differential occurrence would indicate that a fundamental, physiologically relevant, channel property is digitally modulated by bilayer parameters. We will use a stopped-flow bulk assay that measures the quenching of a fluorophore upon Tl+ entry through MthK-reconstituted vesicles to determine the affinity and the kinetics of blockers both in the presence and absence of Ca2+ as well as determine the effect of lipids on desensitization. The proposed aims will provide new insights into the fundamental K+ channel properties, which will be readily applicable to our understanding of the broader family of eukaryotic K+ channels.